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. 2020 Apr 24;23(4):100979.
doi: 10.1016/j.isci.2020.100979. Epub 2020 Mar 12.

Novel Aptamers Selected on Living Cells for Specific Recognition of Triple-Negative Breast Cancer

Simona Camorani  1 , Ilaria Granata  2 , Francesca Collina  3 , Francesco Leonetti  1 , Monica Cantile  3 , Gerardo Botti  3 , Monica Fedele  1 , Mario Rosario Guarracino  2 , Laura Cerchia  4
Affiliations

Novel Aptamers Selected on Living Cells for Specific Recognition of Triple-Negative Breast Cancer

Simona Camorani et al. iScience. .

Abstract

Triple-negative breast cancer (TNBC) is a high heterogeneous group of tumors with a distinctly aggressive nature and high rates of relapse. So far, the lack of any known targetable proteins has not allowed a specific anti-tumor treatment. Therefore, the identification of novel agents for specific TNBC targeting and treatment is desperately needed. Here, by integrating cell-SELEX (Systematic Evolution of Ligands by EXponential enrichment) for the specific recognition of TNBC cells with high-throughput sequencing technology, we identified a panel of 2'-fluoropyrimidine-RNA aptamers binding to TNBC cells and their cisplatin- and doxorubicin-resistant derivatives at low nanomolar affinity. These aptamers distinguish TNBC cells from both non-malignant and non-TNBC breast cancer cells and are able to differentiate TNBC histological specimens. Importantly, they inhibit TNBC cell capacity of growing in vitro as mammospheres, indicating they could also act as anti-tumor agents. Therefore, our newly identified aptamers are a valuable tool for selectively dealing with TNBC.

Keywords: Biochemistry; Cancer; Cell Biology.

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Conflict of interest statement

Declaration of Interests The authors declare no competing interests.

Figures

None
Graphical abstract
Figure 1
Figure 1
TNBC Cell-SELEX (A) Left: schematic protocol for the selection of TNBC-specific aptamers. A pool of 2′F-Py RNAs (contained a 40-mer random sequence region flanked by two constant sequence regions at 5′ and 3′ of 21-mer and 23-mer, respectively) was incubated with BT-474 cells (starting from second round) for a first counterselection step. Unbound sequences were recovered and incubated with MDA-MB-231 cells for the selection step (from second to fourth round) or with A431 cells (from fifth round up to the end of SELEX) for a second counterselection step. Unbound sequences from the second counterselection were recovered and incubated with MDA-MB-231 cells for the selection step. The counterselection was not included in the first round. Unbound sequences were discarded by several washings, and bound sequences were recovered by phenol extraction. Sequences enriched by the selection step were amplified by RT-PCR and in vitro transcribed before a new cycle of selection. Right: characterization of cells used for the SELEX scheme. Lysates from MDA-MB-231, BT-474, and A431 cells were immunoblotted with anti-HER2, anti-EGFR, anti-PR, and anti-ERα antibodies, as indicated. α-tubulin was used as an internal control. (B) Assessment of the selection progress. The indicated selected pools or the TN0 starting library (200-nM final concentration) were incubated for 15 min at 37°C with MDA-MB-231 and BT-474 cells and binding was detected by RT-qPCR. The results are expressed relative to the background binding detected with the TN0 library. Bars depict mean ± SD of three independent experiments.
Figure 2
Figure 2
High-Throughput Sequencing and Data Processing (A) A schematic view of the pipeline used in this study is shown. Briefly, the processing of the raw fastq files involved several steps of filtering after the merging of paired ends, such as quality, length, and frequency filtering. The ranges of the reads number obtained after each step are shown. Finally, 16,500 unique sequences got through the downstream analyses: sequences and secondary structure clustering. The methods used are colored in blue. (B) The plot shows the average frequency (mean ± SD) of the variable region length of the RNA sequences in all the rounds. As expected, the most frequent length is 40 nt. Few sequences show a length of 38, 39, 41, and 42 nt. (C) The plot shows the percentage enrichment at each round (black circle), calculated by: % Enrichment = 1-Unique/Total. A sigmoidal curve fit was added to the plot. (D) Nucleotide frequency distribution for four rounds (0, 3, 9, 14). (E) The heatmap shows the matrix of secondary structure similarities computed by RNAsmc R package. The length of the branch and the colors of the heatmap correspond to degree of similarity between the structures predicted. (F) K-Means clustering of the predicted secondary structures, considering five clusters according to the connectivity parameter. The different clusters are highlighted by the different shape of the points, whereas the color of the ellipses indicates the density of the cluster. The two components explain 66.27% of the point variability. In the boxes are highlighted the candidate sequences, and the arrows indicate their belonging to the specific clusters. (G) Trend of the candidate sequences along the selection rounds. The counts are normalized over the total number of sequences at each round. The y scale is log transformed and the points equal to 0 are not displayed. The candidate aptamers TN1, TN2, and TN3 cover the first three positions according to the slope ranking. They are already present at the round 0 and their concentrations continue to increase till round 14 (TN3) or till round 10 and then show a stable concentration till round 14 (TN1 and TN2). At the round 3 all three of them show a concentration decrease. The candidate aptamers TN20, TN29, TN58, and TN145 start to appear at round 5 (except the TN29 and TN58 that have a weak concentration at round 0) and show an increasing trend till round 14. Sequence 9829 (out of the top 5,000 sequences) is reported as an example of a sequence that did not show up as an enriched sequence in our analysis.
Figure 3
Figure 3
Target-Type Analysis of TNBC Aptamers (A) Following 10-min incubation at RT with 500 nM Alexa 647-labeled TN20 or TN1 aptamers, MDA-MB-231 and BT-474 cells were fixed and incubated with WGA, visualized by confocal microscopy, and photographed. Alexa-labeled aptamer, WGA, and nuclei are visualized in red, green, and blue, respectively. Co-localization results appear yellow in the merged images. All digital images were captured at the same setting to allow direct comparison of staining patterns. (B) Internalization of TN20 into MDA-MB-231 cells. Cells were incubated for 30 min at 37°C in the presence of 500 nM Alexa 647-labeled TN20, after the incubation with LysoTracker to detect acidic organelles inside the cells, visualized by confocal microscopy, and photographed. Alexa-labeled aptamer, WGA, LysoTracker, and nuclei are visualized in red, green, blue, and gray, respectively. Co-localization results appear purple in the merged images. (A and B) Magnification 63×, 0.5× digital zoom, scale bar = 20 μm. Inset: 2× digital zoom, scale bar = 5 μm. (C) Binding of TNBC aptamers to proteinase K-treated MDA-MB-231 (blue) and untreated (red) cells. The concentration of the aptamers in the binding buffer was 200 nM. (A–C) At least three independent experiments were performed.
Figure 4
Figure 4
Targeting Specificity of TNBC Aptamers (A) TNBC aptamers target chemoresistant MDA-MB-231/dox and MDA-MB-231/cis cells with nanomolar Kd values. (B) Binding affinity (1/Kd) of each TNBC aptamer to MDA-MB-231/dox and MDA-MB-231/cis cells is expressed relative to the corresponding binding affinity to MDA-MB-231 cells. Note that the Kd values obtained on parental and chemoresistant cells by using the flow cytometric assay were taken into account for this analysis. (C) Binding of the indicated aptamers (200-nM final concentration, colorimetric assay) on TNBC cells, covering different subtypes (Lehmann et al., 2011), non-TNBC breast cancer cells, human lung fibroblasts (HLF), and MCF10A cells, chosen as non-malignant breast cellular model. The results are expressed relative to the background binding detected with the TN0 negative control. The binding capacity of the aptamers to the cells is reported as ″++″ for high binding (more than 3.5-fold), ″+″ middle binding (between 1.5- and -3.5-fold), and ″−″no binding (less than 1.5-fold). (B and C) Bars depict mean ± SD of three independent experiments. (B) ∗∗p< 0.01; ∗p< 0.05 (one-way ANOVA followed by Tukey's multiple comparison test).
Figure 5
Figure 5
Human Tissue Staining by TNBC Aptamers (A) Images of representative human TNBC cases stained with the six candidate aptamers or TN0 starting library. The images were specifically chosen to represent a variation in staining pattern for each aptamer. Negative/low (left panels) and high (right panels) staining scores are shown. (B) Aptamer-staining scores (calculated based on both staining intensity and cell percentage) on the 18 TNBC samples, a representative triple-positive (ER+, PR+, HER2+) breast cancer, and a representative normal breast sample. Different scores are highlighted by different colors: white (negative, score 0), light blue (low, score 1), dark blue (moderate, score 2–4), and black (high, score 6–9). Groups of samples with same/similar pattern of aptamer staining are marked in red (#6, #10, and #14), in green (#16 and #18), or in purple (#2 and #3). (C) Breast cancer samples expressing ER, PR, and HER2 (upper panels) and breast normal samples dissected adjacent to TNBC cases (lower panels) were stained with TNBC aptamers or TN0 starting library. Images of representative samples of breast cancers and normal tissues stained with a representative TNBC aptamer and TN0 starting library are shown. (A and C) Magnification 20×, scale bar = 100 μm.
Figure 6
Figure 6
TNBC Aptamers Affect Mammosphere-Forming Efficiency of ML TNBC Cells (A and B) (A) MDA-MB-231 and (B) BT-549 cells were plated in ultralow attachment 24-multiwell-plates and grown in stem-permissive conditions in the presence of 400 nM indicated aptamers or TN0 starting library for four days. Representative phase-contrast images are shown. Magnification 10×, scale bar = 250 μm. Cell treatment with specific aptamers, but not the TN0, inhibits both the number, expressed as percentage with respect to mock-treated cells (left), and diameter (right) of mammospheres. (C) RT-qPCR analysis of CD44 gene expression in MDA-MB-231 (left) and BT-549 (right) cells grown in adherent two-dimensional condition (2D) and stem condition in the absence (Mock) or presence of indicated aptamers or TN0 library. (A–C) Bars depict mean ± SD of three independent experiments. ∗∗∗p< 0.001; ∗∗p< 0.01; ∗p< 0.05 relative to mock-treated cells; ##p< 0.01 (unpaired t test). No statistically significant variations among TN0 and mock treatment were obtained.
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